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Proliferating Cell Nuclear Antigen (proliferating + cell_nuclear_antigen)
Selected AbstractsTumour Necrosis Factor Receptor 1 and Hepatocyte Regeneration in Acetaminophen Toxicity: A Kinetic Study of Proliferating Cell Nuclear Antigen and Cytokine ExpressionBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2005Laura P. James To examine the effects of tumour necrosis factor receptor 1 on hepatocyte regeneration, immunohistochemical staining with proliferating cell nuclear antigen was performed. Immunohistochemical staining for proliferating cell nuclear antigen was significantly reduced at multiple time points in the knock-out mice and did not normalize until 96 hr. To evaluate the effect of tumour necrosis factor receptor 1 depletion on cytokines known to be involved in regeneration, levels of macrophage inhibitory protein 2, interferon-,-inducible protein-10 and monocyte chemoattractant protein 1 were compared in the two groups of mice. Significant elevation of all cytokines was observed in both groups of mice; however, higher levels were present in the knock-out mice. Depletion of tumour necrosis factor receptor 1 has long-lasting effects on hepatocyte regeneration in acetaminophen toxicity but multiple other factors appear to orchestrate eventual recovery in these mice. [source] PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNAGENES TO CELLS, Issue 10 2002Tetsuo Iida Background: Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase ,. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied. Results: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA. Conclusion: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp. [source] Chemopreventive effects of rofecoxib and folic acid on gastric carcinogenesis induced by N-methyl-N,-nitro-N-nitrosoguanidine in ratsJOURNAL OF DIGESTIVE DISEASES, Issue 3 2006Su Juan FEI OBJECTIVES: Epidemiological and experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) are chemopreventive agents of gastrointestinal cancers, but few studies on gastric cancer have been carried out. A decrease in folic acid supplement and subsequent DNA hypomethylation are related to gastrointestinal cancers, and it has been shown that high-dose folic acid may interfere with gastric carcinogenesis in dogs. The objective of this study was to investigate the effects of rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, and folic acid on the chemoprevention of gastric cancer induced by N-methyl-N,-nitro-N-nitrosoguanidine (MNNG) in Wistar rats, and to evaluate the cell proliferation of gastric mucosa in different experimental groups. METHODS: Eighty male Wistar rats were randomly divided into five groups (16 rats in each group). In the control group, the rats were given pure water and basal diet. In the MNNG group, the rats received MNNG in drinking water (100 mg/L) and basal diet. In the MNNG + low-dose rofecoxib group, the rats were given MNNG and rofecoxib 5 mg/kg per day with basal diet. In the MNNG + high-dose rofecoxib group, the rats were given MNNG and rofecoxib 15 mg/kg per day with basal diet. In the MNNG + folic acid group, the rats were given MNNG and folic acid 5 mg/kg per day with basal diet. The experiment was terminated at 50 weeks, and all rats were killed. Blood samples of 3 mL were obtained for measurement of serum folic acid concentrations in the control group, the MNNG group and the MNNG + folic acid group by using chemiluminescent method. The stomach was removed from all rats for histopathological examination and immunohistochemical study. Proliferating cell nuclear antigen (PCNA) expression in gastric epithelial cells was also determined. RESULTS: In the MNNG group, five of 11 rats (45.5%) developed gastric cancer, while in all other four groups no gastric cancer was found (P < 0.05). The positivity rate of PCNA expression in the cancerous tissues was significantly higher than that in the non-cancerous tissues (80.0%vs 14.1%, P < 0.05). The positivity rate of PCNA expression in the gastric mucosal cells of the MNNG group was significantly higher than that in the other four groups. The mean serum folic acid concentration of rats was significantly higher in the MNNG + folic acid group (193.70 ± 60.73 ng/mL) than those in the control group (84.21 ± 25.26 ng/mL) and the MNNG group (72.27 ± 16.70 ng/mL, P < 0.05). It was shown that both low- and high-dose rofecoxib as well as folic acid interfered with the development of gastric cancer induced by MNNG in Wistar rats. CONCLUSIONS: The results indicate that rofecoxib as well as folic acid interferes with gastric carcinogenesis induced by MNNG in Wistar rats, and the suppression of gastric cell proliferation may play a crucial role in the chemoprevention of gastric cancer by rofecoxib and folic acid. The higher serum folic acid concentration of rats may play an important role in the prevention of gastric cancer. [source] Recombinant production, crystallization and preliminary X-ray analysis of PCNA from the psychrophilic archaeon Methanococcoides burtonii DSM 6242ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Miranda L. Byrne-Steele Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3,kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5,Å. A single MbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40,Å resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained. [source] Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducensACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Miranda L. Byrne-Steele Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86,Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P63, with unit-cell parameters a = b = 89.0, c = 62.8,Å. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination. [source] Crystallographic study of G178S mutant of human proliferating cell nuclear antigenACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2008Asami Hishiki Proliferating cell nuclear antigen (PCNA) is an evolutionarily conserved protein that forms a ring-shaped homotrimer that functions as a sliding clamp for DNA replication. The rev6-1 mutation of Saccharomyces cerevisiae, which inactivates both translesion DNA synthesis and damage-avoidance pathways while having little effect on normal cell growth, has a G178S substitution in the PCNA protein. Human PCNA protein carrying the G178S substitution was crystallized. Two crystal forms were obtained under similar conditions. Crystal forms I and II belong to space groups P21, with unit-cell parameters a = 84.1, b = 130.2, c = 97.8,Å, , = 113.4°, and P212121, with unit-cell parameters a = 68.1, b = 100.2, c = 131.2,Å, respectively. Structural analyses by molecular replacement are now in progress. [source] Myosin16b: The COOH-tail region directs localization to the nucleus and overexpression delays S-phase progressionCYTOSKELETON, Issue 1 2007Richard S. Cameron Abstract Rat Myo16a and Myo16b comprise the founding members of class XVI myosin and are characterized by an N-terminal ankyrin repeat domain thought to mediate an association with protein phosphatase 1 catalytic subunits 1, and 1,. Myo16b is the principal isoform and reveals predominant expression in developing neural tissue. Here, we use COS-7 cells as a model system to develop an understanding of Myo16b function. We find that Myo16b displays predominant localization in the nucleus of cells transitioning through interphase, but is not associated with processes of mitosis. Using a panel of EGFP-Myo16b-expression plasmids in transient transfection studies, we identified the COOH-terminal residues 1616,1912 as necessary and solely sufficient to target Myo16b to the nucleus. We show that the Myo16b-tail region directs localization to a nuclear compartment containing profilin and polymerized actin, which appears to form a three-dimensional meshwork through the depth of the nucleus. Further, we demonstrate that this compartment localizes within euchromatic regions of the genome and contains proliferating cell nuclear antigen (PCNA) and cyclin A, both markers of S-phase of the cell cycle. Cells transiently expressing Myo16b or Myo16b-tail region show limited incorporation of BrdU, delayed progression through S-phase of the cell cycle, and curtailed cellular proliferation. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2005Yasuhiro Iwao We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity. They divided synchronously regardless of their cell size until they had decreased to 37.5 µm in radius; those smaller than this critical size, however, divided asynchronously with cell cycle times inversely proportional to the square of the cell radius after midblastula transition (MBT). The length of the S phase was determined as the time during which nuclear DNA fluorescence increased in Hoechst-stained blastomeres. When the cell cycle time exceeded 45 min, S and M phases were lengthened; when the cell cycle times exceeded 70 min, the G2 phase appeared; and after cell cycle times became longer than 150 min, the G1 phase appeared. Lengths of G1, S and M phases increased linearly with increasing cell cycle time. Enhanced green fluorescent protein (EGFP)-tagged proliferating cell nuclear antigen (PCNA) expressed in the blastomeres appeared in the S phase nucleus, but suddenly dispersed into the cytoplasm at the M phase. The system developed in this study is useful for examining the cell cycle behavior of the cell cycle-regulating molecules in living Xenopus blastomeres by fluorescence microscopy in real time. [source] Cytologic comparison of a primary parathyroid cancer and its metastatic lesions: A case reportDIAGNOSTIC CYTOPATHOLOGY, Issue 1 2006I.A.C., Katsuhide Ikeda C.T. Abstract We describe the fine-needle aspiration cytology features of a primary parathyroid cancer and of the local recurrent and distant metastatic lesions. The presence of prognostic factors Ki-67 and proliferating cell nuclear antigen (PCNA) was compared immunohistochemically between primary parathyroid carcinoma and related metastatic and recurrent foci. Flow cytometric DNA analysis was also performed to investigate any chromosomal abnormality of the parathyroid carcinoma. Cytologic examination of the endocrine tumor showed that it comprised a loose cohesive cluster and tumor cells with granular cytoplasm and mild nuclear atypia, but for purposes of cytodiagnosis, it is difficult to determine whether such a neoplasm is malignant on the basis of morphology alone. Immunohistochemical analysis showed that Ki-67 and PCNA labeling indices were higher in the recurrent and metastasized carcinomas than in the primary cancer, suggesting that neoplastic cells become more malignant in the recurrent and metastasized foci. To our knowledge, this is the first report describing not only cytopathologic but also immunocytologic differences between primary parathyroid cancer and the metastatic lesion. Diagn. Cytopathol. 2006;34:50-55. © 2005 Wiley-Liss, Inc. [source] mTOR as a potential therapeutic target for treatment of keloids and excessive scarsEXPERIMENTAL DERMATOLOGY, Issue 5 2007C. T. Ong Abstract:, Keloid is a dermal fibroproliferative disorder characterized by excessive deposition of extracellular matrix (ECM) components such as collagen, glycoproteins and fibronectin. The mammalian target of rapamycin (mTOR) is a serine/theronine kinase which plays an important role in the regulation of metabolic processes and translation rates. Published reports have shown mTOR as regulator of collagen expression and its inhibition induces a decrease in ECM deposition. Our aim was to investigate the role of mTOR in keloid pathogenesis and investigate the effect of rapamycin on proliferating cell nuclear antigen (PCNA), cyclin D1, collagen, fibronectin and alpha-smooth muscle actin (, -SMA) expression in normal fibroblasts (NF) and keloid fibroblasts (KF). Tissue extracts obtained from keloid scar demonstrated elevated expression of mTOR, p70KDa S6 kinase (p70S6K) and their activated forms, suggesting an activated state in keloid scars. Serum stimulation highlighted the heightened responsiveness of KF to mitogens and the importance of mTOR and p70S6K during early phase of wound healing. Application of rapamycin to monoculture NF and KF, dose- and time-dependently downregulates the expression of cytoplasmic PCNA, cyclin D1, fibronectin, collagen and , -SMA, demonstrating the anti-proliferative effect and therapeutic potential of rapamycin in the treatment of keloid scars. The inhibitory effect of rapamycin was found to be reversible following recovery in the expression of proteins following the removal of rapamycin from the culture media. These results demonstrate the important role of mTOR in the regulation of cell cycle and the expression of ECM proteins: fibronectin, collagen and , -SMA. [source] Topical treatment with thiazolidinediones, activators of peroxisome proliferator-activated receptor-,, normalizes epidermal homeostasis in a murine hyperproliferative disease modelEXPERIMENTAL DERMATOLOGY, Issue 3 2006Marianne Demerjian Abstract:, In a murine model of epidermal hyperplasia reproducing some of the abnormalities of several common skin disorders, we previously demonstrated the antiproliferative and pro-differentiating effects of peroxisome proliferator-activated receptor (PPAR),, PPAR,/,, and liver X receptor activators. Unlike other subgroups of PPAR activators, thiazolidinediones (TZDs), a family of PPAR, ligands, did not inhibit keratinocyte proliferation in normal murine skin. Here, we studied the effects of two TZDs, namely ciglitazone (10 mM) and troglitazone (1 mM), in the same murine model where epidermal hyperproliferation was reproduced by repeated barrier abrogation with tape stripping. Topical treatment with ciglitazone and troglitazone resulted in a marked and significant decrease in epidermal thickness. Furthermore, in all TZD-treated groups, we observed a significant decrease in keratinocyte proliferation using proliferating cell nuclear antigen, 5-bromo-2,-deoxyuridine, and tritiated thymidine incorporation. However, using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, we found no difference in apoptosis between different treatments, emphasizing that it is the antiproliferative role of these activators that accounts for the decrease of epidermal thickness. Finally, using immunohistochemical methods, we determined the effects of ciglitazone on keratinocyte differentiation in this hyperproliferative model. We observed an increased expression of involucrin and filaggrin following ciglitazone treatment, suggesting a pro-differentiating action of TZDs in this model. In summary, topical TZDs significantly reduce epidermal keratinocyte proliferation while promoting differentiation in a murine model of hyperproliferative epidermis. Together, these results suggest that in addition to their metabolic effects currently in use in the treatment of type 2 diabetes, topical TZDs could be considered as potential alternative therapeutic agents in hyperproliferative skin diseases such as psoriasis. [source] Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1 , a molecular link between recombination and chromatin assembly during meiosisFEBS JOURNAL, Issue 9 2008Satomi Ishii In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. ,-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1,PCNA complex in meiotic events. We propose that the CAF-1,PCNA complex modulates chromatin assembly following meiotic recombination. [source] Re-oxygenation of hypoxic simian virus 40 (SV40)-infected CV1 cells causes distinct changes of SV40 minichromosome-associated replication proteinsFEBS JOURNAL, Issue 9 2002Hans-Jörg Riedinger Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the,origin region. After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place. To,further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation. T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase ,, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia. The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase ,, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation. Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells. T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase. The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work. [source] The reconstituted human Chl12-RFC complex functions as a second PCNA loaderGENES TO CELLS, Issue 4 2004Yasushi Shiomi The sister chromatid cohesion factor Chl12 shares amino acid sequence similarity with RFC1, the largest subunit of replication factor C (RFC), and forms a clamp loader complex in association with the RFC small subunits RFCs2-5. It has been shown that the human Chl12-RFC complex, reconstituted with a baculovirus expression system, specifically interacts with human proliferating cell nuclear antigen (PCNA). The purified Chl12-RFC complex is structurally indistinguishable from RFC, as shown by electron microscopy, and it exhibits DNA-stimulated ATPase activity that is further enhanced by PCNA, and by DNA binding activity on specific primer/template DNA structures. Furthermore, the complex loads PCNA onto a circular DNA substrate, and stimulates DNA polymerase , DNA synthesis on a primed M13 single-stranded template in the presence of purified replication proteins. However, it cannot substitute for RFC in promoting simian virus 40 DNA replication in vitro with crude fractions. These results demonstrate that the human Chl12-RFC complex is a second PCNA loader and that its roles in replication are clearly distinguishable from those of RFC. [source] Central role for Cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extractsGENES TO CELLS, Issue 6 2000Satoru Mimura Background In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood. Results Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase , and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase , onto chromatin required Cdc45 but did not require DNA polymerase ,. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase , on chromatin. Conclusions These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA. [source] Synovial chondromatosis: the possible role of FGF 9 and FGF receptor 3 in its pathologyINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2000Dror Robinson Primary synovial chondromatosis (PSC) is a rare disorder of the synovium typified by cartilaginous nodule formation within the synovial membrane. Fibroblast growth factor receptor 3 (FGFR3) is a recently described specific marker of mesenchymal precartilaginous stem cells. Expression patterns of FGFR3 and its specific ligand, fibroblast growth factor 9 (FGF 9), were evaluated both in situ and in cell cultures. Histologically, cells at the periphery of the cartilage nodules express FGFR3 and PCNA ( proliferating cell nuclear antigen). Elevated levels of FGF 9, its specific ligand, have been found in synovial fluids of patients with synovial chondromatosis. Synoviocytes but not chondrocytes from affected patients express FGF9 in culture. This pattern is absent in normal synovium and cartilage. Downregulation of FGF9 may provide a possible nonoperative therapy for PSC. [source] Cell proliferation and differentiation during fracture healing are influenced by locally applied IGF-I and TGF-,1: Comparison of two proliferation markers, PCNA and BrdUJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2003B. Wildemann Abstract Growth factors IGF-I and TGF-,1 are known to stimulate fracture healing. The purpose of this study was to investigate the role of locally applied IGF-I and TGF-,1 during the early phase of fracture healing (Days 5, 10, and 15 after fracture) on cellular processes like proliferation and differentiation in a rat model. Two different immunohistochemical markers were used to analyze cell proliferation: (1) injection of the thymidine analogue BrdU and subsequent immunohistochemical staining for BrdU-positive nuclei, and (2) the antibody against the "proliferating cell nuclear antigen" (PCNA). In comparison, both methods revealed similar results concerning the types of proliferating cells at the different time points and the two groups. Labeling indices of both methods showed very good correlation (e.g., rs: 0.887 and p < 0.001 at day 10 in the control group without growth factors). Comparison of the callus morphology and the proliferation rate showed differences during fracture healing due to the local application of IGF-I and TGF-,1 from coated implants. At Day 5 the callus of the group treated with growth factors displayed an earlier appearance of cartilage compared to the control group. This was accompanied by an onset of cell proliferation in chondrocytes. Likewise, at the later time points an enhanced maturation of the callus tissue and the proliferation pattern were detectable in the growth-factor group. These results indicate that local application of IGF-I and TGF-,1 accelerates early cellular processes during fracture healing. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 65B: 150,156, 2003 [source] Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP-,AGING CELL, Issue 3 2010Simone A. Van Den Berge Summary A main neurogenic niche in the adult human brain is the subventricular zone (SVZ). Recent data suggest that the progenitors that are born in the human SVZ migrate via the rostral migratory stream (RMS) towards the olfactory bulb (OB), similar to what has been observed in other mammals. A subpopulation of astrocytes in the SVZ specifically expresses an assembly-compromised isoform of the intermediate filament protein glial fibrillary acidic protein (GFAP-,). To further define the phenotype of these GFAP-, expressing cells and to determine whether these cells are present throughout the human subventricular neurogenic system, we analysed SVZ, RMS and OB sections of 14 aged brain donors (ages 74-93). GFAP-, was expressed in the SVZ along the ventricle, in the RMS and in the OB. The GFAP-, cells in the SVZ co-expressed the neural stem cell (NSC) marker nestin and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Mcm2. Furthermore, BrdU retention was found in GFAP-, positive cells in the SVZ. In the RMS, GFAP-, was expressed in the glial net surrounding the neuroblasts. In the OB, GFAP-, positive cells co-expressed PCNA. We also showed that GFAP-, cells are present in neurosphere cultures that were derived from SVZ precursors, isolated postmortem from four brain donors (ages 63-91). Taken together, our findings show that GFAP-, is expressed in an astrocytic subpopulation in the SVZ, the RMS and the OB. Importantly, we provide the first evidence that GFAP-, is specifically expressed in longterm quiescent cells in the human SVZ, which are reminiscent of NSCs. [source] Development of granulocytes in haematopoietic tissues of Rhizoprionodon lalandiiJOURNAL OF FISH BIOLOGY, Issue 4 2002F. J. Pacheco Granulocytes of the epigonal and Leydig organs of Rhizoprionodon lalandii were identified and classified into three different cell types, type I and type II eosinophils and neutrophils. The development of these cells in the haematopoietic tissues was dynamic, demonstrated by nuclear immunopositivity for the proliferating cell nuclear antigen (PCNA) proteins and was regulated by various cytokines, including the transforming growth factor , -l (TGF/,1). The expression pattern of these cells was heterogeneous among individual cells and TGF/,1 -immunostaining was found principally in the cytoplasm of immature granulocytes. The presence of TGF/,1 in cells about to divide was demonstrated suggesting that modulation of differentiation and proliferation occurs in the haematopoietic tissues of this species of elasmobranch. [source] Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phaseJOURNAL OF NEUROCHEMISTRY, Issue 1 2002Christopher L. Bacon Abstract Cell cycle progression is tightly regulated by cyclins, cyclin-dependent kinases (cdks) and related inhibitory phophatases. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and mapped the temporal regulation of selected cyclins, cdks and inhibitory proteins throughout the 12 h of G1 by immunoblot analysis. The D-type cyclins, D3 and D1, were differentially expressed during the C6 glioma G1 phase. Cyclin D1 was up-regulated in the mid-G1 phase (4,6 h) while cyclin D3 expression emerged only in late G1 (9,12 h). The influence of the anticonvulsant agent valproic acid (VPA) on expression of cyclins and related proteins was determined, since its teratogenic potency has been linked to cell cycle arrest in the mid-G1 phase. Exposure of C6 glioma to VPA induced a marked up-regulation of cyclin D3 and decreased expression of the proliferating cell nuclear antigen. In synchronized cell populations, increased expression of cyclin D3 by VPA was detected in the mid-G1 phase (3,5 h). Immunocytochemical localization demonstrated rapid intracellular translocation of cyclin D3 to the nucleus following VPA exposure, suggesting that VPA-induced cell cycle arrest may be mediated by precocious activation of cyclin D3 in the G1 phase. [source] Smoking delays chondrogenesis in a mouse model of closed tibial fracture healingJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 12 2006Hossam B. El-Zawawy Abstract Smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders, including long bone fracture. In the current study, a murine model of tibial fracture healing was used to test the hypothesis that smoking delays chondrogenesis after fracture. Mice were divided into two groups, a nonsmoking control group and a group exposed to cigarette smoke for 1 month prior to surgical tibial fracture. Mice were euthanized at 7, 14, and 28 days after surgery. The outcomes measured were immunohistochemical staining for type II collagen protein expression as a marker of cartilage matrix and proliferating cell nuclear antigen (PCNA) staining to measure proliferation at the site of injury. Toluidine blue staining and histomorphometry were used to quantify areas of cartilaginous and noncartilaginous fracture callus. Radiographs were analyzed for evidence of remodeling after injury. At day 7 after injury, mice exposed to cigarette smoke had a smaller fracture callus with less cartilage matrix compared to controls. Proliferation was present at high levels in both groups at this time point, but proliferating cells had a more immature morphology in the smoking group. At day 14, chondrogenesis was more active in smokers compared to controls, while a higher percentage of bone was present in the control animals. At day 28, X-ray analysis revealed a larger fracture callus remaining in the smoking animals. Together, these findings show that the chondrogenic phase of tibial fracture healing is delayed by smoking. This study represents, to our knowledge, the first analysis of molecular and cellular mechanisms of healing in a smoking mouse fracture model. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Effect of ketoprofen in topical formulation on vascular endothelial growth factor expression and tumor growth in nude mice with osteosarcomaJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2004Kenshi Sakayama Abstract OST cells, a low metastatic cell line established from human osteosarcoma, were inoculated under the periosteum of the ossa cranii of nude mice. Four weeks later, tumors were percutaneously treated for an additional 4 weeks with a patch containing either placebo or ketoprofen (KP). In the placebo group, OST cells formed osteoid and invaded the cranial bone. Tumor mass weighed 3.54 g. Approximately 85% of cells within the tumor expressed proliferating cell nuclear antigen (PCNA), indicating that they were proliferating with a high mitotic activity. Many feeder vessels were located within the tumor. The majority of tumor cells expressed intensely vascular endothelial growth factor (VEGF). In the KP group, invasion of OST cells into the cranial bone was suppressed and the tumor mass was 47% of that of the placebo group. Approximately 65% of cells within the tumor were PCNA-negative, indicating that their growth was arrested. There were considerably fewer feeder vessels within the tumor in the KP group than in the placebo group. Only a small number of cells expressed VEGF. Based on these findings, we concluded that topical administration of KP to nude mice with osteosarcoma inhibited VEGF expression, reduced the development of feeder vessels for supply of nutrients and oxygen, and suppressed tumor growth. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] In vitro and in vivo cytokeratin patterns of expression in bioengineered human periodontal mucosaJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2009I. Garzón Background and Objective:, Development of human oral mucosa substitutes by tissue engineering may provide new therapeutic tools for the management of periodontal diseases. In this study we evaluated a fibrin,agarose human oral mucosa substitute both in vitro and in vivo. Material and Methods:,In vitro bioengineered oral mucosa substitutes were developed from irrelevant biopsy samples of human oral gingiva. In vivo evaluation of the constructed tissues was performed by implantation into athymic nude mice. The expression of several epithelial markers was assessed by microarray analysis and immunohistochemistry. Results:, Bioengineered oral mucosa samples kept in vitro developed a multilayered epithelium that expressed several cytokeratins, including some markers of simple epithelia (cytokeratins 7, 8 and 18), along with markers of stratified epithelia (cytokeratins 5 and 13) and of cell proliferation (proliferating cell nuclear antigen). Bioengineered tissues grafted in vivo onto nude mice exhibited very good biointegration with the host, showing a cytokeratin expression pattern that was very similar to that of normal native oral mucosa controls. Histological analysis of the artificial tissues demonstrated that oral mucosa substitutes evaluated in vivo were structurally mature, showing some typical structures of human native oral mucosa such as rete ridges and chorial papillae, along with numerous blood vessels at the fibrin,agarose stromal substitute. These structures were absent in samples evaluated in vitro. Conclusion:, The results indicate that this model of human oral mucosa, constructed using fibrin,agarose scaffolds, shows similarities to native oral mucosa controls and imply that bioengineered oral mucosa substitutes could eventually be used clinically. [source] Histomorphometric characteristics and expression of epidermal growth factor and its receptor by epithelial cells of normal gingiva and hereditary gingival fibromatosisJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2003C. S. A. Araujo Objective:, The objective of this study was to examine the histomorphometric features and evaluate the expression of epidermal growth factor (EGF) and transmembranic receptor (EGFr) and the proliferative potential of epithelial cells from normal and hereditary gingival fibromatosis (HGF) gingival tissues. Background:, EGF is a multifunctional cytokine with a variety of biological effects including stimulation of cell proliferation by binding to its specific EGFr. Methods:, Immunohistochemistry was performed to measure EGF and EGFr expression and the epithelial cell proliferation was determined by measuring proliferating cell nuclear antigen (PCNA). Results:, Histomorphometric evaluation indicated that in HGF the mean height of the epithelial papillae was higher compared to the normal gingiva (NG), whereas mean epithelial area and number of epithelial papillae were quite similar in both groups. The EGF and EGFr positive cells were observed in the basal, spinous and granular cell layers of both normal and HGF tissues, with a gradual reduction from the basal layer. Although the expressions of EGF and EGFr in the control group were significantly higher than those from HGF, in HGF the epithelial papilla tips showed increased number of proliferating cells and elevated expression of EGF and EGFr. There was a correlation between the proliferative potential of epithelial cells and the expression of EGF or EGFr only in the epithelial papilla tips of HGF gingiva. Conclusion:, Our data suggest that EGF and EGFr in the oral epithelium of HGF gingiva may stimulate epithelial cell proliferation, with the resultant apical migration of the oral epithelium and formation of the slender deep epithelial papillae; however, without hyperplastic alterations. [source] Bioavailability and efficacy of antisense morpholino oligomers targeted to c- myc and cytochrome P-450 3A2 following oral administration in ratsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2002Vikram Arora Abstract Antisense phosphorodiamidate Morpholino oligomers (PMO) are resistant to degradation by cellular hydrolases, DNases, RNases, and phosphodiesterases, but remain sensitive to prolonged exposure to low pH. The present studies evaluate the oral fractional bioavailability, stability, and efficacy of two distinct PMO sequences targeted to c- myc and cytochrome P-450 (CYP) 3A2. The c- myc antisense 20-mer, AVI-4126 (5,-ACGTTGAGGGGCATCGTCGC-3,), slowed the regenerative process in the rat liver after a 70% partial hepatectomy (PH). Rats were administered 3.0 mg/kg AVI-4126 in 0.1 mL saline via a bolus intravenous injection or in 0.5 mL sterile phosphate-buffered saline via gavage immediately following PH. The areas under the plasma concentration versus time curves revealed a fractional oral availability of 78.8% over a period of 10 min through 24 h. Immunoblot analysis of liver tissue from rats treated orally with AVI-4126 demonstrated a sequence-specific reduction in the target protein c-Myc, as well as secondary proliferation markers: proliferating cell nuclear antigen (PCNA), cyclin D1, and p53. The CYP3A2 antisense 22-mer AVI-4472 (5,-GAGCTGAAAGCAGGTCCATCCC-3,) caused a sequence-dependent reduction of approximately five-fold in the rat liver CYP3A2 protein levels and erythromycin demethylation activity in 24 h following oral administration at a dose of 2 mg/kg. It is concluded that oral administration of PMOs can inhibit c- myc and CYP3A2 gene expression in rat liver by an antisense-based mechanism of action. These studies highlight the potential for development of PMOs as orally administered therapeutic agents. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1009,1018, 2002 [source] Effects of isorhynchophylline on angiotensin II-induced proliferation in rat vascular smooth muscle cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2008Feng Zhang Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in cardiovascular diseases. Isorhynchophylline, an alkaloid from a traditional Chinese medicine Gambirplant, has been used to treat cardiovascular diseases. The aim of this study was to investigate the effects of isorhynchophylline on angiotensin II (Ang II)-induced proliferation of rat VSMCs. VSMCs were isolated from rat artery and cultured for 14 days before experimentation. The effect of isorhynchophylline on Ang II-induced proliferation was evaluated by cell number, MTT assay and flow cytometry, and nitric oxide (NO) content and activity of NO synthase (NOS) were measured. The expression of proto-oncogene c-fos, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) mRNAs was measured by real-time RT-PCR. VSMC cultures were verified by morphology and immunostaining with ,-smooth muscle actin. Isorhynchophylline (0.1,10.0 ,M) was not toxic to VSMCs, but markedly decreased Ang II (1.0 ,m)-enhanced cell number and MTT intensity, and blocked cell transition from G0/G1 to S phase. Furthermore, isorhynchophylline increased the NO content and NOS activity, and suppressed Ang II-induced over-expression of c-fos, OPN and PCNA. Thus, isorhynchophylline was effective against Ang-II induced cell proliferation, an effect that appears to be due, at least in part, to increased NO production, regulation of the cell cycle, and depressed expression of c-fos, OPN and PCNA related to VMSC proliferation. [source] Lack of evidence that bone marrow cells contribute to cholangiocyte repopulation during experimental cholestatic ductal hyperplasiaLIVER INTERNATIONAL, Issue 4 2006Yuki Moritoki Abstract: Background: Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear. Methods: Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%,-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated. Results: Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7. Conclusions: In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model. [source] Selective elimination of hepatic natural killer T cells with Concanavalin A improves liver regeneration in miceLIVER INTERNATIONAL, Issue 3 2006Wen Huang Abstract: Background: Although concanavalin A (Con A) as a T cell stimulant can cause natural killer T (NKT) cell-mediated liver injury in mice and a nonhepatotoxic dose of Con A can trigger innate immune cells including NKT cells to prevent tumor metastasis in the liver, little is known about the role of Con A-primed NKT cells in liver repair. In this study, we aimed to investigate the effect of pretreatment with a nontoxic dose of Con A on subsequent liver regeneration in mice. Methods: A nontoxic dose of Con A was injected intravenously 24 h before partial hepatectomy (PHx), which was used as a model of liver regeneration. Ratios of remnant liver mass to body weight, bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) labeling were used to assess liver regeneration. Results: Hepatic mononuclear cells were isolated and analyzed by flow cytometry. After PHx, the ratios of liver weight to body weight, PCNA-positive hepatocytes and BrdU-positive hepatocytes in Con A-pretreated mice were significantly higher than that of phosphate-buffered saline-treated mice, indicating that Con A pretreatment can accelerate liver regeneration. Flow cytometric analysis showed that NKT cells were significantly activated and selectively eliminated after the Con A administration. Moreover, NKT cells expressed more apoptosis-related molecules, Fas and Annexin V. Conclusions: Taken together, Con A accelerates liver regeneration in mice by eliminating hepatic NKT cells via activation-induced cell death. [source] Epigenetic reprogramming of liver cells in tamoxifen-induced rat hepatocarcinogenesisMOLECULAR CARCINOGENESIS, Issue 3 2007Volodymyr P. Tryndyak Abstract Tamoxifen, a nonsteroidal anti-estrogen, is a potent genotoxic hepatocarcinogen in rats, with both tumor initiating and promoting properties. Recently it has been demonstrated that genotoxic carcinogens, in addition to exerting genotoxic effects, often cause epigenetic alterations and these induced epigenetic changes may play important mechanistic role in carcinogenesis. In the present study, we investigated the role of tamoxifen-induced epigenetic changes in hepatocarcinogenic process. The results of the study showed that exposure of female F344 rats to tamoxifen resulted in progressive loss of CpG methylation in regulatory sequences of long interspersed nucleotide elements (LINE-1) and prominent increase in expression of LINE-1 elements and c- myc proto-oncogene. The accumulation of tamoxifen-induced DNA lesions was accompanied by the decreased level of Rad51, Ku70, and DNA polymerase , (Pol,) proteins that play a crucial role in maintenance of genomic stability. Furthermore, feeding rats with tamoxifen-containing diet led to increased regenerative cell proliferation, as indicated by the increased level of Ki-67 and proliferating cell nuclear antigen (PCNA) proteins. These data indicate that exposure of animals to genotoxic hepatocarcinogen tamoxifen led to early phenotypical alterations in livers characterized by emergence of epigenetically reprogrammed cells with a specific cancer-related epigenetic phenotype prior to tumor formation. © 2006 Wiley-Liss, Inc. [source] Tyrosine phosphorylation of tau accompanies disease progression in transgenic mouse models of tauopathyNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 6 2010K. Bhaskar K. Bhaskar, G. A. Hobbs, S-H. Yen and G. Lee (2010) Neuropathology and Applied Neurobiology36, 462,477 Tyrosine phosphorylation of tau accompanies disease progression in transgenic mouse models of tauopathy Aim: Tau protein is a prominent component of paired helical filaments in Alzheimer's disease (AD) and other tauopathies. While the abnormal phosphorylation of tau on serine and threonine has been well established in the disease process, its phosphorylation on tyrosine has only recently been described. We previously showed that the Src family non-receptor tyrosine kinases (SFKs) Fyn and Src phosphorylate tau on Tyr18 and that phospho-Tyr18-tau was present in AD brain. In this study, we have investigated the appearance of phospho-Tyr18-tau, activated SFK and proliferating cell nuclear antigen (PCNA) during disease progression in a mouse model of human tauopathy. Methods: We have used JNPL3, which expresses human tau with P301L mutation, and antibodies specific for phospho-Tyr18-tau (9G3), ser/thr phosphorylated tau (AT8), activated SFK and PCNA. Antibody staining was viewed by either epifluorescence or confocal microscopy. Results: Phospho-Tyr18-tau appeared concurrently with AT8-reactive tau as early as 4 months in JNPL3. Some 9G3-positive cells also contained activated SFKs and PCNA. We also investigated the triple transgenic mouse model of AD and found that unlike the JNPL3 model, the appearance of 9G3 reactivity did not coincide with AT8 in the hippocampus, suggesting that the presence of APP/presenilin influences tau phosphorylation. Also, Thioflavin S-positive plaques were 9G3-negative, suggesting that phospho-Tyr18-tau is absent from the dystrophic neurites of the mouse triple transgenic brain. Conclusions: Our results provide evidence for the association of tyrosine-phosphorylated tau with mechanisms of neuropathogenesis and indicate that SFK activation and cell cycle activation are also involved in JNPL3. [source] | |||||||||||||||||||||||||||||||